Metabolic signalling to the ovary

卵巢的代谢信号

• 批准号: RGPIN-2014-03956
• 负责人: Duggavathi, Raj
• 金额: $ 2.19万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=649723
• 关键词: Metabolic; signalling; ovary

Financial support is sought for the continuation of my NSERC research program with a long-term goal of understanding the mechanisms metabolic signaling to the ovary. The short-term goal for the present period of funding is to understand the role of insulin like growth factor 1 (IGF1) system in the regulation of ovarian function. Specific objectives are: 1) To explore the importance of the IGF1 of liver and granulosa cell origin; 2) to determine the mechanisms of IGF1 receptor in FSH signalling; and 3) to decipher molecular mechanisms by which gonadotropins regulate the expression of PAPPA in bovine granulosa cells **RATIONALE: Infertility in lactating dairy cattle has been attributed to severe negative energy balance due to high metabolic demands of milk production. Abnormal levels of metabolic hormone such as insulin, leptin and IGF1 are the major feature negative energy balance. Our recent data demonstrate that circulating concentrations of IGF1 are dramatically reduced in lactating cows as compared to heifers. This abnormal endocrine milieu is reflected in the follicular fluid of the dominant follicles of lactating cows as compared to those at similar developmental stage in heifers. Reduced follicular fluid IGF1 concentration is associated with decreased expression of PAPPA in granulosa cells of the dominant follicle in lactating cows. As PAPPA is a protease of IGF1 binding proteins, these data indicate that the follicular microenvironment of lactating cows is severely deficient of bioactive IGF1. Thus, a better understanding of the molecular mechanisms of IGF1 in ovarian follicles will contribute toward development of novel treatment and/or management strategies to improve fertility in lactating dairy cows.**For objective one, we propose to develop liver- and granulosa-specific Igf1 knockout mice using our expertise in cre-recombinase directed deletion of genes. For this we will breed mice carrying conditional allele of Igf1 with mice expressing Cre-recombinase specifically in the liver (Alb1-Cre) and granulosa cells (Cyp19-Cre), respectively. We will undertake extensive phenotyping approaches to determine consequences of loss of Igf1 production in the liver or granulosa cells on ovarian follicular dynamics, reproductive hormonal profile and fertility. For the second objective, we will use a well-established culture system of bovine primary granulosa cells to investigate the signaling pathways of IGF1R in the regulation of granulosa cell proliferation and steroidogenesis. We will employ gene knockdown and pharmacologic inhibition strategies to test signaling pathways of IGF1R in bovine granulosa cells. For the third objective, we will first employ an in vivo ultrasound guided follicular aspiration strategy to synchronize follicular wave and collect samples. We will systematically analyze expression pattern of PAPPA, its abundance in follicular fluid and its protease activity in follicles at specific developmental stages. We will then investigate the molecular mechanisms by which FSH regulates the expression of PAPPA in bovine granulosa cells using cutting edge molecular biology techniques. Our aim will be to determine signaling pathways and transcription factors involved in FSH-regulated expression of PAPPA.**Overall, the results of the proposed research program should significantly enhance our understanding how IGF1 system regulates ovarian functions and thus female fertility.

为了继续我的NSERC研究计划，寻求财政支持，长期目标是了解卵巢代谢信号的机制。本基金的短期目标是了解胰岛素样生长因子1（IGF 1）系统在卵巢功能调节中的作用。具体目标是：1）探索肝脏和颗粒细胞来源的IGF 1的重要性; 2）确定IGF 1受体在FSH信号传导中的机制; 3）解读促性腺激素调节牛颗粒细胞中PAPPA表达的分子机制 ** 依据：泌乳奶牛的不孕症归因于产奶的高代谢需求导致的严重负能量平衡。代谢激素如胰岛素、瘦素和IGF 1水平异常是能量负平衡的主要特征。我们最近的数据表明，IGF 1的循环浓度显着降低泌乳奶牛相比，小母牛。与处于相似发育阶段的小母牛相比，泌乳奶牛优势卵泡的卵泡液反映了这种异常的内分泌环境。卵泡液IGF 1浓度降低与泌乳奶牛优势卵泡颗粒细胞PAPPA表达降低相关。由于PAPPA是IGF 1结合蛋白的蛋白酶，这些数据表明泌乳奶牛的卵泡微环境严重缺乏生物活性IGF 1。因此，更好地了解IGF 1在卵泡中的分子机制将有助于开发新的治疗和/或管理策略，以提高泌乳奶牛的生育力。对于目标之一，我们建议开发肝脏和粒细胞特异性IgF 1基因敲除小鼠，利用我们的专业知识，在基因重组酶定向删除。为此，我们将分别在肝脏（Alb 1-Cre）和颗粒细胞（Cyp 19-Cre）中特异性表达Cre重组酶的小鼠与携带Igf 1条件等位基因的小鼠交配。我们将进行广泛的表型分析方法，以确定肝脏或颗粒细胞中Igf 1产生的损失对卵巢卵泡动力学，生殖激素谱和生育能力的影响。对于第二个目标，我们将使用一个完善的培养系统，牛原代颗粒细胞，研究IGF 1 R的信号通路在调节颗粒细胞增殖和类固醇激素合成。我们将采用基因敲除和药理学抑制策略来测试牛颗粒细胞中IGF 1 R的信号通路。对于第三个目标，我们将首先采用体内超声引导卵泡抽吸策略来同步卵泡波并收集样本。我们将系统地分析PAPPA的表达模式，其在卵泡液中的丰度和其蛋白酶活性在特定发育阶段的卵泡。然后，我们将研究FSH调节PAPPA在牛颗粒细胞中的表达的分子机制，使用尖端的分子生物学技术。我们的目标是确定参与FSH调节PAPPA表达的信号通路和转录因子。**总的来说，拟议的研究计划的结果应该显着提高我们的理解IGF 1系统如何调节卵巢功能，从而女性生育能力。